DNA methylation and chromatin structure of the constitutively expressed normal and amplified human dihydrofolate reductase (DHFR) gene were studied. Only the 5' promoter region of the DHFR gene was found to be undermethylated, while the remaining 30 kilobase (kb) gene was completely methylated. The promoter region was DNasel hypersensitive in chromatin. In methotrexate (MTX) resistant HeLa cells with an amplified DHFR gene, all copies of the amplified gene exhibited a pattern of unermethylation and DNaseI hypersensitivity of only the promoter region. Detailed mapping of the DAaseI hypersensitive sites revealed five discrete cutting sites; two were also sensitive to S1 nicking when this DNA fragment was part of supercoiled plasmid DNA. DNA mediated gene transfer showed that in vitro methylation of the promoter region markedly reduced transformation frequency of DHFR- CHO cells. The DHFR gene fragment in cells having the DHFR+ phenotype had become specifically demethylated in the promoter region. Chromosomal organization of the DHFR gene family was studied by DNA analysis of human X rodent somatic cell hybrids. The functional gene and five intronless pseudogenes were found to be dispersed to different chromosomes. The functional gene was assigned to chromosome 5 and two pseudogenes, hDHFR psi2 and psi4 to chromosomes 6 and 3, respectively. A intronless pseudogene with perfect sequence homology to the functional locus (hDHFR-psi1) was found to be polymorphic in that it is present in DNA from some individuals but not others.